The lysosphingolipid, sphingosine 1-phosphate (S1P) is carried in the blood in association with lipoproteins, predominantly high density lipoproteins (HDL). Emerging evidence indicates that many of the cardiovascular effects of HDL may be attributable to its S1P cargo. We have evaluated how levels of S1P and related sphingolipids in the HDL-containing fraction of human serum correlate with occurrence of ischemic heart disease (IHD). To accomplish this we used liquid chromatography-mass spectrometry to measure S1P levels in an HDL containing fraction of serum (depleted of LDL and VLDL) from 204 subjects in the Copenhagen City Heart Study (CCHS). The study group consisted of individuals having high serum HDL cholesterol (HDL-C) (females:e 73.5 mg/dL;males:e 61.9mg/dL) and verified IHD;subjects with high HDL-C and no IHD;individuals with low HDL-C (females:d 38.7 mg/dL;males:d 34.1 mg/dL) and IHD, and subjects with low HDL-C and no IHD. The results show a highly significant inverse relationship between the level of S1P in the HDL-containing fraction of serum and the occurrence of IHD. Furthermore, a similar inverse relationship with IHD was also observed for two other sphingolipids, dihydro-S1P and C24:1-ceramide, in the HDL- containing fraction of serum. These findings indicate that compositional differences in sphingolipid content of HDL might be important in deciphering the putative protective role of HDL in IHD. To extend on our preliminary findings we will measure S1P levels in HDLs from CCHS serum samples to determine the prognostic value of HDL-S1P levels in assessing the relative risk for IHD. Given that our previous studies do not preclude that S1P associated with another plasma S1P carrier, albumin, might also correlate with occurrence of IHD, we plan to determine whether S1P levels associated with albumin inversely correlate with occurrence of IHD. To gain mechanistic insights, we intend to measure the impact of low serum levels of lipoprotein-associated S1P on the process of atherosclerosis in a mouse model. In addition, we will test the hypothesis that HDLs containing reduced levels of S1P are dysfunctional with respect to their potential to promote cardioprotective signaling. To accomplish this we will evaluate the ability of HDLs from CCHS subjects with defined levels of S1P to promote endothelial barrier activity and activate the Map kinase and Akt signaling pathways. PUBLIC HEALTH RELEVANCE: While evidence indicates that high blood levels of HDL are cardioprotective, there are individuals with very high levels of HDL that have heart disease suggesting that qualitative differences might exist in HDL particles which make them functionally different with respect to cardioprotection. We have discovered that individuals with high levels of HDL and clinical evidence of heart disease have low levels of sphingosine-1-phosphate (S1P), a lipid normally carried on HDL that has been shown to have a number of beneficial effects on the cardiovascular system. In this application, experimentation will be performed to test the hypothesis that HDL-associated S1P levels inversely correlate with cardiovascular disease risk and to determine whether HDL particles with lower than normal levels of S1P are dysfunctional with respect to cardioprotective-related activities.